当归多糖组分AP-3诱生小鼠脾细胞IL-2和IFN-γ的作用

目的研究当归多糖组分AP-3对细胞因子IL-2和IFN-γ的诱生作用，以探讨其免疫调节的特点。方法 流式细胞术测定培养的脾细胞中CD4⁺细胞比例；酶联免疫法测定培养上清液中IL-2和IFN-γ的浓度；RT-PCR法测定IL-2和IFN-γ mRNA的转录水平。结果AP-3在0.6～2 μmol·L^-1，能显著提高培养脾细胞CD4⁺细胞的百分率；在2～6 μmol·L^-1，AP-3时间、剂量依赖性地增加培养的细胞上清液中IL-2的浓度和细胞内IL-2 mRNA的转录水平，而对于IFN-γ和IFN-γ mRNA，则先升高后降低，并呈现剂量依赖性。结论当归多糖能够促进IL-2和IFN-γ的分泌，激活Th1细胞，从而发挥免疫调节作用。     

拟人参皂苷F11在大鼠体内的药物代谢研究

目的探讨拟人参皂苷F11在大鼠体内的药物代谢产物及其过程.方法ip拟人参皂苷F₁₁后,应用TLC分析排泄物中的代谢产物,并利用制备薄层分离制备代谢产物,通过波谱解析(MS,¹HNMR,¹³CNMR,¹H-¹HCOSY)确定其结构.结果从粪便中分离鉴定了3种代谢产物,分别为拟人参皂苷R_T5,ocotillol和1个新的代谢产物F-3-1,并确定其结构为6-O-α-L-吡喃鼠李糖基(1-2)-β-D-吡喃葡糖基-(20S,23S,24R)-达玛-20(24)-环氧-3β,6α,12β,23,25-五醇(6-O-α-L-rhamnopyranosyl-(1-2)-β-D-glucopyranosyl-(20S,23S,24R)-dammar-20(24)-epoxy-3-β,6α,12β,23,25-pentanol).但在尿液和胆汁中并未发现任何代谢产物.结论拟人参皂苷F₁₁不被肝脏代谢,但胆汁排泄物可在肠道被代谢为水解和氧化产物.     

基于Keap1/Nrf2/HO-1通路汉黄芩素7-O-β-D-乙基葡萄糖醛酸苷体外抗氧化应激作用研究

目的 探讨汉黄芩素 7-O-β-D-乙基葡萄糖醛酸苷（WODE）对脂多糖（LPS）诱导的小鼠巨噬细胞（RAW264.7）的抗氧化应激作用及机制。方法 MTS法检测WODE（2.5、5.0、10.0、20.0、40.0、80.0、160.0 μmol·L^-1）对RAW264.7细胞活力的影响；体外培养RAW264.7细胞，WODE（10、20、40 μmol·L^-1）或地塞米松（1 μmol·L^-1，阳性药）预处理1 h，再给予LPS刺激24 h（造模过程不再加药），对照组不加 LPS 和受试物，模型组只给予 LPS 刺激；荧光探针检测胞内活性氧（ROS）水平；Griess反应测定细胞上清液中 NO 生成量；ELISA 检测细胞上清液中肿瘤坏死因子-α（TNF-α）和白细胞介素-6（IL-6）的分泌；实时荧光定量 PCR（qRT-PCR）法检测细胞内诱导型一氧化氮合成酶（iNOS）、环氧化酶-2（COX-2）、白细胞介素-1β（IL-1β）、醌氧化还原酶1（NQO-1）、超氧化物歧化酶-1（SOD-1）的mRNA表达水平；Western blotting法检测细胞核因子E2相关因子2（Nrf2）和血红素加氧酶-（1 HO-1）蛋白表达水平；免疫荧光染色法检测细胞内Kelch ECH相关蛋白（1 Keap1）表达水平。结果 与对照组比较，WODE浓度小于40 μmol·L^-1时，细胞存活率没有明显变化；浓度大于80 μmol·L^-1时，细胞存活率下降，但未见统计学差异。与模型组比较，WODE 10、20、40 μmol·L^-1组 ROS水平显著降低（P＜0.01）；20、40 μmol·L^-1 组 NO 释放显著降低（P＜0.05、0.01）；40 μmol·L^-1 组 iNOS mRNA 表 达 水 平 显 著 降 低（P＜0.01）；10、20、40 μmol·L^-1 组 COX-2 和20、40 μmol·L^-1组IL-1β mRNA表达水平显著降低（P＜0.05、0.01）；10、20、40 μmol·L^-1组TNF-α和IL-6的释放受到显著抑制（P＜0.01）；10、20、40 μmol·L^-1组NQO-1 mRNA表达水平显著升高（P＜0.01），40 μmol·L^-1组SOD-1 mRNA表达水平显著升高（P＜0.01）；10、20、40 μmol·L^-1组Keap1蛋白表达水平显著降低（P＜0.01）；10、20、40 μmol·L^-1组HO-1蛋白和mRNA表达水平显著提高（P＜0.05、0.01）；20、40 μmol·L^-1 组 Nrf2 蛋白和 40 μmol·L^-1 组 Nrf2 mRNA 表达水平显著增加（P＜0.01）。结论 WODE 对 LPS 诱导的RAW264.7细胞的氧化应激具有抑制作用，其作用机制可能与调控Keap1/Nrf2/HO-1信号通路相关。     

人参皂苷Rb1抑制β淀粉样蛋白25-35诱导的皮层神经元tau蛋白过度磷酸化

目的探讨人参皂苷Rb1对凝聚态β-AP_25-35诱导的胎鼠皮层神经元tau蛋白过度磷酸化的影响及其可能的作用机制。方法通过蛋白免疫印迹法和免疫细胞化学染色法检测神经元tau蛋白磷酸化水平、总tau蛋白水平和糖原合成酶3β(GSK-3β)的蛋白表达水平。结果凝聚态β-AP_25-35(20 μmol·L^-1)作用于皮层神经元12 h，tau蛋白磷酸化水平和总tau蛋白水平均增高，同时GSK-3β蛋白表达也增多。用人参皂苷Rb1或GSK-3β特异性抑制剂氯化锂预处理后，凝聚态β-AP_25-35诱导的tau蛋白的过度磷酸化受到明显抑制，同时GSK-3β的表达也降低。结论人参皂苷Rb1可通过抑制GSK-3β的表达来抑制凝聚态β-AP_25-35诱导的皮层神经元tau蛋白的过度磷酸化。     

五味子酚对氧自由基损伤小鼠脾淋巴细胞的保护作用

研究了五味子酚(Sal)对氧自由基损伤小鼠脾淋巴细胞的影响。体外实验结果表明,Sal在5×10^-6mol·L^-1时对Fe²⁺-VitC引起的脾淋巴细胞GSH含量降低有明显的抑制作用,且能阻抑Fe²⁺-Cys引起的MDA生成增加,改善细胞膜的流动性。用扫描电镜观察到Sal在5×10^-4mol·L^-1时可逆转Fe²⁺-VitC引起的脾淋巴细胞表面微绒毛皱折减少、细胞变形等病理改变。体内高氧分压应激损伤小鼠实验表明,igSal20mg·kg^-1×8d可逆转脾淋巴细胞SOD活性代偿性增高,并提高脾淋巴细胞内GSH含量。以上结果提示Sal对氧自由基损伤脾淋巴细胞有保护作用。     

段木栽培及袋栽灵芝多糖对体外培养小鼠脾淋巴细胞增殖活性的比较

目的 比较段木栽培灵芝多糖(wood-cultured Ganoderma lucidum polysaccharides, GL-PS-WC) 及袋栽灵芝多糖(bag-cultured Ganoderma lucidum polysaccharides, GL-PS-BC)对体外培养小鼠脾淋巴细胞增殖活性的影响,探讨袋栽灵芝多糖替代段木栽培灵芝多糖的可能性。 方法检测两种灵芝多糖对混合淋巴细胞培养(MLC)反应的影响;观察对刀豆蛋白A (Con A)、细菌脂多糖(LPS)诱导淋巴细胞增殖的影响以及对环孢素A (CsA)、丝裂霉素C(Mit C)、足叶乙苷(VP-16) 等抑制MLC反应的影响。结果当质量浓度为0.2～12.8 mg·L^-1时,两种灵芝多糖均可促进MLC反应,增强Con A或LPS诱导的淋巴细胞增殖,并拮抗CsA, Mit C或VP-16对MLC反应的抑制作用。未发现两种多糖之间有显著性差异。结论GL-PS-WC及GL-PS-BC对体外培养脾淋巴细胞的增殖活性有类似作用。     

重组人胸腺素α原体外对IFN-γ,IFN-α及TNF-α的影响

目的体外观察重组人胸腺素α原(prothymosin α,Pro Tα)对几种重要细胞因子分泌的影响。方法用脾淋巴细胞、脾巨噬细胞及腹腔巨噬细胞,以ELISA法检测Pro Tα对IFN-γ,IFN-α和TNF-α分泌的影响。结果1×10^-7 mol·L^-1 Pro Tα明显促进脾细胞分泌IFN-γ(P<0.05),该浓度的Pro Tα也明显刺激小鼠脾巨噬细胞分泌IFN-α(P<0.01);在小鼠腹腔巨噬细胞中,Pro Tα能明显刺激IFN-α和TNF-α的分泌(P<0.01)。结论Pro Tα对细胞因子IFN-γ,IFN-α和TNF-α的分泌均有促进作用。     

人参皂苷Rb1可能通过CDK5途径减轻Aβ25-35诱导的胎鼠海马神经元tau蛋白过度磷酸化

观察人参皂苷Rb1对Aβ_25-35诱导的海马神经元tau蛋白过度磷酸化的影响，并探讨其对周期依赖性蛋白激酶(cyclin-dependent kinase 5，CDK5)及激动亚基p25/p35的可能作用。通过蛋白免疫印迹法和免疫细胞化学染色法检测胎鼠海马神经元tau蛋白在Thr²⁰⁵、Ser³⁹⁶和Ser⁴⁰⁴位点的磷酸化水平，及CDK5的两个亚基cdk5和p25/p35的蛋白水平。20 μmol·L^-1凝聚态Aβ_25-35作用于海马神经元12 h，可使海马神经元tau蛋白在Thr²⁰⁵、Ser³⁹⁶和Ser⁴⁰⁴位点的磷酸化水平增高，p25的数量增多，但并不影响cdk5亚基的表达。人参皂苷Rb1可减轻凝聚态Aβ_25-35诱导的海马神经元tau蛋白的过度磷酸化，抑制p35的降解并减少海马神经元p25的生成。人参皂苷Rb1可能通过CDK5途径减轻Aβ_25-35诱导的胎鼠海马神经元tau蛋白过度磷酸化。     

人参皂苷Rb1通过JNK/p38 MAPK途径减轻Aβ25-35诱导的胎鼠皮层神经元tau蛋白过度磷酸化

探讨在Aβ_25-35(beta-amyloid peptide (25-35)，Aβ_25-35)诱导的拟阿尔茨海默病样胎鼠皮层神经元tau蛋白过度磷酸化中，人参皂苷Rb1对tau蛋白磷酸化及JNK/p38 MAPK的可能作用。应用蛋白免疫印迹和免疫细胞化学染色的方法，观察tau蛋白磷酸化和JNK(c-jun N-terminal kinase)/p38 MAPK的表达情况。凝聚态Aβ_25-35(20 μmol·L^-1)作用于皮层神经元12 h，tau蛋白的磷酸化水平明显增高，同时JNK/p38 MAPK的总量及其活性形式——磷酸化JNK/p38 MAPK的蛋白表达水平也增加，人参皂苷Rb1可以减轻tau蛋白的磷酸化水平及JNK/p38 MAPK的蛋白水平。人参皂苷Rb1可通过JNK/p38 MAPK途径减轻Aβ_25-35诱导的tau蛋白过度磷酸化。     

丁基苯酞对原代培养胎大鼠皮层神经细胞外液NO及胞浆内cGMP水平的影响

用分光光度法和放射免疫分析法分别检测NO及cGMP水平,同时观察l-丁基苯酞(l-NBP)和d-丁基苯酞(d-NBP)对原代培养的胎大鼠皮层神经细胞外液NO及胞浆内cGMP水平的影响。结果表明,d-NBP(0.1～100μmol·L^-1)对低糖低氧条件下或含N-甲基-D-门冬氨酸(NMDA)或含KCl的培养基中皮层神经细胞外液NO及细胞内cGMP水平明显升高,而l-NBP(0.1～100μmol·L^-1)则能明显降低NO和cGMP水平。提示:d-NBP和l-NBP对低糖低氧,NMDA或KCl诱导的NO释放和cGMP生成有相反的作用。     

Protopanaxatriol-type ginsenosides differentially modulate type 1 and type 2 cytokines production from murine splenocytes

Seven protopanaxatriol-type ginsenosides and their aglycones including PPT, PT, -Re, -Rg (1), -F (1), -Rh (1), 20(R)-Rh (1) which are closely related in structure were studied for their effects on type 1 and type 2 cytokines production from murine splenocytes and their related mechanisms were examined. The results indicate that PPT, PT and ginsenoside-Re show hardly any or weak effects on concanavalin A (Con A)-induced production of IFN-gamma and IL-4. Ginsenoside-Rh (1) and 20(R)-Rh (1) induce a Con A-induced type 1 cytokine pattern by increasing the production of interleukin-12 (IL-12), the expression of IFN-gamma, T-bet and enhancing NF-kappaB DNA binding activity. In contrast ginsenosides-Rg (1) and -F (1) cause a Con A-induced type 2 cytokines response by increasing the expression of IL-4, GATA-3 and enhancing NF-kappaB DNA binding activity. Thus, these protopanaxatriol-type ginsenosides have different immunomodulatory effects, which might explain the complex immunomodulatory effect of Panax ginseng.     

Immunoregulatory abnormalities of T cells and hyperreactivity of B cells in the<Emphasis Type="Italic">In Vitro</Emphasis> immune response in pristane-induced lupus mice

Systemic lupus erythematosus (SLE) is characterized by overactive B cells that differentiate into autoantibody-forming cells, aberrant T cell function that provides helping B cells produce autoantibodies, and overproduction of proinflammatory cytokines. However, immunodysregulation in lupus pathogenensis remains incomplete. We examined mitogen-stimulated production of proinflammatory cytokines, cell proliferation, T cell activation, and T cell apoptosis in vitro in pristane-induced lupus BALB/c mice compared to normal mice. LPS-stimulated production of IL-6 and IL-10 by splenocytes and macrophages from pristane-induced lupus mice were remarkably up-regulated compared to normal mice, whereas production of macrophage TNF-alpha was significantly down-regulated. Moreover, in vitro production of IL-2, IL-6, IL-10 and IFN-gamma by Con A-stimulated splenocytes, cell proliferation in LPS- or Con A-stimulated- thymocytes and splenocytes, and expression of CD69+CD4+ T cells in Con A-stimulated splenocytes were greatly increased in cells derived from pristane-induced lupus mice compared to normal mice. In addition, splenic T cells and CD4+ T cells in thymocytes from pristane-induced lupus mice were more resistant than nonautoimmune normal cells to Con A-induced apoptosis. Our findings indicate that immunoregulatory abnormalities of T cells and hyperreactivity of B cells in the in vitro immune responses in pristane-induced lupus mice may explain some of lupus pathogenesis.     

Carbofuran suppresses T-cell-mediated immune responses by the suppression of T-cell responsiveness, the differential inhibition of cytokine production, and NO production in macrophages

The effects of carbofuran (2,3-dihydro-2,2-dimethyl-7-benzo-furanol N-methylcarbamate) on the functions of T cells in splenocytes and peritoneal macrophages were examined in view of T-cell-mediated immune response (CMIR) in male C57BL/6 mice. Intraperitoneal administration of carbofuran (0.075, 0.15 and 0.3 mg/kg body weight) resulted in significant suppression of delayed type hypersensitivity (DTH), indicating that it caused the suppression of CMIR. Carbofuran decreased Concanavalin A (Con A)- and alloantigen-induced proliferation, and interleukin (IL)-2 production of splenocytes. In vitro addition of rIL-2 could not completely restore the suppressed T-cell proliferation, and IL-2-induced proliferation of Con A-activated splenocytes was also suppressed, which implied that carbofuran caused defects in IL-2 production and responsiveness of splenocytes to IL-2, leading to the suppression of T-cell proliferation. Con A-induced production of interferon-gamma (IFN-gamma) was significantly suppressed by carbofuran, while that of IL-4 was not affected. The production of transforming growth factor-beta from splenocytes was also significantly inhibited by carbofuran. Judging from these results, carbofuran might directly suppress the cytokine production in T helper 1 (Th1) cells. In addition, IFN-gamma-induced production of nitric oxide (NO) in macrophages was also inhibited by carbofuran, which might be one of the important mechanisms of carbofuran-induced CMIR suppression in mice. Collectively, the present study suggests that carbofuran might suppress CMIR through the suppression of T-cell responsiveness, IFN-gamma production in Th1 cells, and NO generation in macrophages.     

Comparative study of the endotoxemia and endotoxin tolerance on the production of Th cytokines and macrophage interleukin-6: differential regulation of indomethacin

Endotoxin tolerance reduces the capacity of monocytes to produce proinflammatory cytokines, results in cellular immune paralysis, and down-regulates the production of helper T (Th)1 type cytokines with a shift toward a Th2 cytokine response. Prostaglandin (PG)E2 in the immune system also results in macrophage inactivation and the suppression of Th1 activation and the enhancement of Th2 activation. However, the inhibitory effects of PGE2 on the altered polarization of the Th cell and macrophage interleukin (IL)-6 production characterized in part by cellular immune paralysis in a state of endotoxin tolerance is unclear. This study was undertaken, using indomethacin, to investigate the role of endogenous PGE2 on the Th cytokines and macrophage IL-6 production in a state of endotoxin tolerance compared to those with endotoxemia mice, wherein, in this latter case, the increased production of proinflammatory cytokines and PGE2 is exhibited. Endotoxemia was induced by injection of lipopolysaccharide (LPS; 10 mg/kg in saline) ip. once in BALB/c mice, and endotoxin tolerance was induced by pretreatment with LPS (1 mg/kg in saline) injected i.p. daily for two consecutive days and then with LPS 10 mg/kg on day 4. Splenocytes or macrophages were obtained from endotoxemia and endotoxin tolerance models pretreated with indomethacin, and then cytokine production was induced by Con A-stimulated splenocytes for the Th cytokine assays and LPS-stimulated macrophages for the IL-6 assay. Our results showed that endotoxemia led to significantly reduced IL-2 and IL-4 production, to significantly increased IL-6 production, whereas interferon (IFN)-gamma production was not affected. Indomethacin in the case of endotoxemia markedly attenuated IFN-gamma and IL-6 production and didnt reverse IL-2 and IL-4 production. Endotoxin tolerance resulted in the significantly reduced production of IL-2 and IFN-gamma and the significantly increased production of IL-4 and IL-6. Indomethacin in endotoxin tolerance greatly augmented IL-2 production, significantly decreased IL-4 production, and slightly attenuated IL-6 production. These findings indicate that endogenous PGE2 may mediate the suppressed Th1 type immune response, with a shift toward a Th2 cytokine response in a state of endotoxin tolerance, whereas endotoxemia may be regulated differentially. Also, endogenous PGE2 may mediate macrophage IL-6 production in the case of endotoxemia to a greater extent than in the case of endotoxin tolerance.     

Curcumin regulated shift from Th1 to Th2 in trinitrobenzene sulphonic acid-induced chronic colitis

AIM: To investigate the therapeutic effects of curcumin (Cur) on trinitrobenzene sulphonic acid (TNBS)-induced colitis and the effects of Cur on the balance of Th1/Th2 cytokines. METHODS: Colitis was induced by TNBS and treated with Cur (30 mg/kg/d, ip), dexamethasone (Dex, 2 mg/kg/d), or Cur plus dexamethasone (Cur+Dex, 30 mg/kg/d Cur ip+2 mg/kg/d Dex,ip). mRNA in colon mucosa were detected by real-time quantitative polymerase chain reaction. Intracellular cytokines were detected by flow cytometry and concentrations of cytokines in sera were detected by enzyme-linked immunosorbent analysis. RESULTS: Both Cur and Dex improved body weight loss, ameliorated histological images and decreased macroscopic score and myeloperoxidase activity. Cur decreased the expression of Th1 cytokines (IL-12, IFN-gamma, TNF-alpha, IL-1) and increased the expression of Th2 cytokines (IL-4 and IL-10) in colon mucosa. Cur also increased the proportion of IFN-gamma/IL-4 in splenocytes and circulation. Dex and Cur+Dex decreased the expression of Th1 cytokines but could not increase the expression of Th2 cytokines and the proportion of IFN-gamma/IL-4. CONCLUSION: Cur exerted therapeutic effects on colitis by regulating the shift from Th1 to Th2.     

A newly devised formulation for self-medication enhances interferon-gamma production and proliferation of splenic lymphocytes

A newly devised formulation for self-medication in Toyama, PanaWang, is a new herbal medicine (so called Toyama original brand formulation) developed based on traditional philosophy and scientific evidence. We here tried to examine the effect of oral administration of PanaWang on the balance of type I helper T cells (Th1) and Th2 cells. Splenic lymphocytes from normal mice were stimulated with Concanavalin A (Con A) in vitro and the secretion of Th1- and Th2-type cytokines, interferon-gamma (IFN-gamma) and interleukin-4 (IL-4) respectively, was investigated. Con A-induced production of IFN-gamma from spleen cells, but not IL-4, was enhanced by the administration of PanaWang. Increased production of IFN-gamma was also detected in splenic lymphocytes from Th2-predominant BALB/c mice after DNP-immunization, without a change in antigen-specific IgE levels in vivo. Antigen-specific proliferative responses were also increased in lymphocytes from PanaWang-treated mice. These findings raise the possibility that PanaWang has Th1-stimulating activity and induces Th1-predominant immunity.     

Antidepressants suppress production of the Th1 cytokine interferon-gamma, independent of monoamine transporter blockade.

In this study, antidepressants with selectivity for the noradrenaline transporter (reboxetine and desipramine), or the serotonin transporter (fluoxetine and clomipramine) were examined in terms of their ability to promote an anti-inflammatory cytokine phenotype in human blood. In addition, we examined the ability of trimipramine; a tricyclic antidepressant that is devoid of monoamine reuptake inhibitory properties on cytokine production. Lipopolysaccharide (LPS) was used to stimulate monocyte-derived pro-inflammatory (IL-1beta, TNF-alpha, IL-12) and anti-inflammatory (IL-10) cytokines, whilst concanavalin A (Con A) was used to stimulate T-cell (Th(1): IFN-gamma and Th(2/3): IL-10) cytokines. All of the antidepressants suppressed IFN-gamma production in the 10-50 microM concentration range, irrespective of their preference for serotonin or noradrenaline transporters. This suppression of IFN-gamma production was paralleled by reduced T-cell proliferation, therefore we suggest that the ability of antidepressants to suppress IFN-gamma production may be related to their anti-proliferative properties. The fact that trimipramine also suppressed IFN-gamma production and T-cell proliferation indicates that these immunomodulatory actions of antidepressants are most likely unrelated to inhibition of monoamine reuptake. Interestingly, exposure to a lower concentration (1 microM) of the antidepressants tended to increase T-cell-derived IL-10 production, with significant effects elicited by the noradrenaline reuptake inhibitors reboxetine and desipramine. In contrast to the robust actions of antidepressants on T-cell derived cytokine production, they failed to induce any consistent change in LPS-induced monocyte cytokine production. Overall, our results indicate that IFN-gamma producing T-cells (Th(1) cells) are the major target for the immunomodulatory actions of antidepressants, and provide evidence questioning the relationship between the monoaminergic reuptake properties of antidepressants and their immunomodulatory effects. The potential clinical significance of the anti-inflammatory actions of antidepressants is discussed.     

Berberine down-regulates the Th1/Th2 cytokine gene expression ratio in mouse primary splenocytes in the absence or presence of lipopolysaccharide in a preventive manner

Berberine is a natural isoquinoline alkaloid. This study investigated the effects of berberine on cytokine gene expression in mouse primary splenocytes in the absence or presence of lipopolysaccharide (LPS) using 4 different experimental models in vitro. The relative expression of the following cytokine genes was determined using a real-time quantitative polymerase chain reaction assay: pro-inflammatory tumor necrosis factor (TNF)-α, anti-inflammatory interleukin (IL)-10, T-helper type 1 (Th1) (IL-2), and Th2 (IL-4) cytokines. The results showed that berberine down-regulated ratios of the relative Th1 (IL-2)/Th2 (IL-4) cytokines expression fold in mouse primary splenocytes in the absence or presence of LPS in a preventive manner. This study suggests that berberine may possess anti-inflammatory potential by shifting the Th1/Th2 balance toward Th2 polarization.